Genetic testing results

Click on the picture above to read about the upcoming event in London. There are familiar faces appearing, like Agnieszka, Dr Boles and indeed me.

 

I am quite often sent genetic testing results. There are many types of tests ranging from inexpensive tests looking at SNPs to the expensive WES or WGS tests.

SNP = Snip = Single Nucleotide Polymorphism = a tiny genetic spelling mistake

WES = Whole Exome Sequencing

WGS = Whole Genome Sequencing

There is a small industry based around selling expensive supplements for SNPs.

We all carry thousands of SNPs and I think these tests may often raise issues that are not causal.  The results from WGS or WES can be much more insightful.  A good example being in the comment recently posted on this blog.

 

I've been following your blog for many years, it's a real blessing and the perfect place to come and read for us, parents of ASD kids. My boy, 9, has non-regressive autism, is largely non verbal (one word sentence) and has pronounced OCD symptoms (similar to excoriating disorder, but aimed at the environment), hyperactivity and severe gut problems, recurrent vomitting, gastroparesis, etc. The only thing that visibly stopped the hyperactivity and inappropriate laughing and helped him sit for longer periods of time and read his books or watch whole movies was 0.5/kg mg Naltrexone daily, as advised by this paper https://pubmed.ncbi.nlm.nih.gov/16735648/. Lower doses saw the OCD creep back. As for his WGS test results, I've found relevant the fact that he has four pathogenic mutations in the EIF4EBP1, also a de novo mutation in the PIK3R1 gene and multiple other mutations in the STAT3, HTR3a, MAPT and also HLA-DRB1, HLA-DQA1, HLA-A, HLA-B, HLA-C, NRG1, NRG2, SCN4a, CACNA1S genes, amongst many others. We recently tried a course of Azythromycin for immuno-modulation, which saw his OCD reduced further, also his academic interest and focus increased visibly. He responds very well to Ibuprofen, AlkaSeltzer gold, Propranolol, Sytrinol and Cromolyn, but a quite long trial of Bumetanide two years ago did nothing for him. After all trials of various protocols and individual drugs, his gut is still bad, very often food seems to have major difficulty to pass though his digestive tract, no matter how finely tuned his diet is or how many prokinetics he takes. Given your extensive knowledge, I've always wondered what your take on the underlying problem/genetic pathway might be in his case (microglial activation, MTOR activation, perhaps?) and what drugs/cocktail of drugs might work best for his specific genetics and symptoms. He is a smart boy, has self-taught reading, loves music and masters his iPAD like a pro and, unlike what we know about autism, loves being around people. I cannot give up on him. We live in the UK, not the best place to even talk about treatments for autism. Please, if it's not too much to ask, tell me what other medications you thing it might boost his cognition further and help him start talking and develop more skills. Sorry for the long post. And thank you for any advice and ideas you might have to offer.

 

It would be useful to know which of the above mutations are present in at least one of the parents.  There so many possibly causal mutations here; I expect some are actually not relevant. In other words, it is not as scary at it may appear to be.

I do like to start with the easy part, which will be the ion channels.  Dysfunctions in ion channels (channelopathies) are often treatable with existing drugs and there is a great deal of information on each one.

 

CACNA1S

This gene encodes the calcium channel Cav1.1.

This is known as an L type calcium channel, the other ones being Cav1.2 and Cav1,3 and Cav1.4.

These ion channels are extremely important to how your brain works.  Because they also play a role in how your heart works, numerous drugs have been developed, some are more specific to one type of channel (Amlodipine for Cav1.3, Verapamil for Cav1.2).

The individual channels interact with other sub-types, so a mutation in one sub-type can affect other subtypes.

Very interesting in this case are the GI problems. There were efforts made a few years ago to develop R-verapamil as a drug to treat IBS/IBD under the name of Rezular. Some readers of this blog have reported that the only thing that resolves their child’s GI problems is an L-type calcium channel blocker.

Note Memantine, which is an Alzheimer’s drug that was subject to a very large autism clinical trial in the US.  The trial was deemed a failure, but one reader told me that Memantine is the only drug she had found that solved her child’s GI problems.  Memantine has several different modes of action, and a little reported one is blocking L-type calcium channels.

 

https://www.mdpi.com/1648-9144/49/9/64

Conclusions. Our results suggest that the neuroprotective effect of memantine could arise not only through the inhibition of the NMDA receptor current but also through the suppression of the L-type Ca²⁺ current.   

 

You might expect/hope a geneticist would suggest treatment with a drug like Verapamil.

  

SCN4a

This gene encodes the sodium ion channel Nav1.4.

This is one of the genes associated with Hypokalemic Periodic Paralysis (HPP), that was covered extensively in this blog. Interestingly the above Cav1.1 is also associated with Hypokalemic Periodic Paralysis (HPP).

The other genetic cause of HPP is KCNJ2 (an inward-rectifier potassium channel Kir2.1).

The immediate recovery therapy is drinking a potassium supplement.

A common preventative measure is acetazolamide (Diamox). This drug has also been covered in previous posts. The proposed mechanism is that it “increases the flow of potassium” – not sure what that is supposed to mean.

Some common anti-epilepsy drugs block Nav1.4 (Lamotrigine, Phenytoin etc).

All of the above-mentioned drugs have been used in autism. In specific cases they have shown a benefit.

You could ask your doctor to cautiously try them one by one.

Interestingly, the drug that seems to help many with sound sensitivity is Ponstan.  This cheap drug that affects the flow of potassium ions was proposed by Knut Witkowski as a therapy for 2-3 year olds to prevent non-verbal severe autism. 

 

EIF4EBP1

Here you mention there are 4 pathogenic mutations.

This gene is a real mouthful, but regular reader might recall the odd looking eIF4E part appearing in some previous posts

“This gene encodes one member of a family of translation repressor proteins. The protein directly interacts with eukaryotic translation initiation factor 4E (eIF4E), which is a limiting component of the multi subunit complex that recruits 40S ribosomal subunits to the 5' end of mRNAs. Interaction of this protein with eIF4E inhibits complex assembly and represses translation. This protein is phosphorylated in response to various signals including UV irradiation and insulin signaling, resulting in its dissociation from eIF4E and activation of cap-dependent mRNA translation.”

eIF4E inhibitors for Autism – Why not Ribavirin?

 

As you can see in the above post there are numerous ways to block elF4E. It is possible that the 4 mutations in your gene EIF4EBP1 could have the reverse effect in which case you would want to activate elF4E, not block it.

On the list, in my post above, is quercetin which is OTC and simple to try.

 

PIK3R1

A mutation in this gene can alter the PI3K/AKT/mTOR signaling pathway.

If this gene is causing a problem you might see some facial features a triangular face, a prominent forehead, small chin with a dimple, a loss of fat under the skin, prominent ears, hearing loss and delayed speech.

A mutation in this gene can lead to SHORT syndrome, which hopefully your pediatrician will have heard of.

 https://rarediseases.info.nih.gov/diseases/7633/short-syndrome

 

STAT3

STAT3 plays a key role in the immune system and elsewhere.

You can either have too much or too little STAT3.

In lay terms the immune system might end up either over-activated (hence benefiting from Ibuprofen and Cromolyn sodium) or under activated.

The immunomodulatory probiotics prescribed by gastroenterologists might be worth a try.

Lactobacillus rhamnosus GG

Lactobacillus plantarum 299v 

 

This might well reduce GI problems as well.

  

HTR3a 

This gene encodes subunit A of the type 3 serotonin receptor. It has lots of effects, but it may contribute to the vomiting.

It is associated with:

• Motion sickness
• Irritable bowel syndrome
• Social phobia
• Serotonin syndrome

For gastroparesis (impaired stomach's motility) the good drug seems to be Domperidone, which you should be able to get for free from your NHS doctor.

Another very popular therapy for gut dysbiosis of all kinds in some countries, but not the UK, is sodium butyrate. This has been mentioned in previous posts. It is an OTC supplement that will produce butyric acid in the gut and it helps restore a healthy mucosa. If you eat lots of fiber and have a healthy microbiome you would produce butyric acid naturally. The cheapest place in Europe to buy it is Poland, where they sell a product called Intesta Max (a weaker version is Intesta).  In the UK it is 3 times more expensive. Making friends with a Pole will save you money.

 

MAPT

The MAPT gene makes tau proteins.  There is a class of disease called tauopathy.

Tau Reduction Prevents Key Features of Autism in Mouse Models

 

Tau: A Novel Entry Point for mTOR-Based Treatments in Autism Spectrum Disorder?

 

As with the PIK3R1 mutation this will lead you to the idea of targeting mTOR signalling. You can inhibit this with Rapamycin, which has been used in autism.

 

Rapamycin/Sirolimus Improves the Behavior of an 8-Year-Old Boy With Nonsyndromic Autism Spectrum Disorder

 

One UK reader did get Everolimus prescribed on the NHS, but that was because the child was diagnosed with a genetic disorder called TSC. Several readers of this blog have tried Rapamycin as used in the Chinese case study.

If you do not have an over activated immune system, Rapamycin will cause the problem of an underactive immune system.

  

HLA-DRB1, HLA-DQA1, HLA-A, HLA-B, HLA-C,

 These genes all play a role in the immune system.

The human leukocyte antigen (HLA) system is a complex of genes in humans which encode cell-surface proteins responsible for regulation of the immune system.

The immune system uses the HLAs to differentiate self cells and non-self cells. Any cell displaying that person's HLA type belongs to that person and is therefore not an invader.

 

HLA Immune Function Genes in Autism

The human leukocyte antigen (HLA) genes on chromosome 6 are instrumental in many innate and adaptive immune responses. The HLA genes/haplotypes can also be involved in immune dysfunction and autoimmune diseases. It is now becoming apparent that many of the non-antigen-presenting HLA genes make significant contributions to autoimmune diseases. Interestingly, it has been reported that autism subjects often have associations with HLA genes/haplotypes, suggesting an underlying dysregulation of the immune system mediated by HLA genes. Genetic studies have only succeeded in identifying autism-causing genes in a small number of subjects suggesting that the genome has not been adequately interrogated. Close examination of the HLA region in autism has been relatively ignored, largely due to extraordinary genetic complexity. It is our proposition that genetic polymorphisms in the HLA region, especially in the non-antigen-presenting regions, may be important in the etiology of autism in certain subjects.

One specific HLA gene has been studied in autism.

 Inheritance of HLA-Cw7 Associated With Autism Spectrum Disorder (ASD)

Autism spectrum disorder (ASD) is a behaviorally defined disorder that is now thought to affect approximately 1 in 69 children in the United States. In most cases, the etiology is unknown, but several studies point to the interaction of genetic predisposition with environmental factors. The immune system is thought to have a causative role in ASD, and specific studies have implicated T lymphocytes, monocytes, natural killer (NK) cells, and certain cytokines. The human leukocyte antigen (HLA) system is involved in the underlying process for shaping an individual’s immune system, and specific HLA alleles are associated with specific diseases as risk factors. In this study, we determine whether a specific HLA allele was associated with ASD in a large cohort of patients with ASD. Identifying such an association could help in the identification of immune system components which may have a causative role in specific cohorts of patients with ASD who share similar specific clinical features. Specimens from 143 patients with ASD were analyzed with respect to race and ethnicity. Overall, HLA-Cw7 was present in a much greater frequency than expected in individuals with ASD as compared to the general population. Further, the cohort of patients who express HLA-Cw7 shares specific immune system/inflammatory clinical features including being more likely to have allergies, food intolerances, and chronic sinusitis as compared to those with ASD who did not express HLA-Cw7. HLA-Cw7 has a role in stimulating NK cells. Thus, this finding may indicate that chronic over-activation of NK cells may have a role in the manifestation of ASD in a cohort of patients with increased immune system/inflammatory features.

 

The therapeutic implication would be to look at immunomodulatory therapy.

At the simple level you have NSAIDs like Ibuprofen, but then you have the more potent drugs used to treat psoriasis, arthritis, IBD etc.

If you saw Dr Arthur Krigsman, the autism gastroenterologist, I guess he would prescribe Humira.  This is an injection you take every few weeks.  That very well might help your son in many ways. He does also come to Europe for consultations. You would need a colonoscopy.

Some British parents take their autistic kids with GI problems to Italy for treatment. You could ask the Thinking Autism charity who they go to see. One of these doctors presented at their conference in London in 2019.  He used some of Krigsman’s slides in his presentation.

 

NRG1, NRG2

Neuregulin 1 and 2 are implicated in brain disorders. NRG1 is well known as a schizophrenia gene, but it has been shown to be miss-expressed in autism as well.

NRG2 also plays a role in many neurological conditions.  

Neuregulins in Neurodegenerative Diseases 

The downstream effect of NRG1 is on epidermal growth factor (EGF). There are expensive cancer drugs like Lapatinib that are inhibitors of EGFR. 

As I have written in my blog, disturbed growth factors is a recurring feature of autism. This is why son many autism genes are also cancer genes. Don’t worry, this does not mean everyone with autism is going to get cancer.

 

Conclusion

Try and find a doctor who is interested to treat your son.

I think you will make great strides by treating the GI problems that you see every day.

I did meet an UK autism mother at that conference in London in 2019 who was told by her doctor that her son’s GI problems would not be treated in the UK and she should look abroad. She went to Italy and solved his problems.  It sounds so bizarre, I would not have believed it to be possible, had I not been talking directly to the mother.  I did talk to the Italian gastroenterologist at that same event.  Contact Thinking Autism and ask who was the Italian who presented in 2019.

Gene Primer for people interested in Autism

Today’s post is a standalone introduction to genetics, as relevant for lay people interested in autism.

The scientists among you will likely know all this and more, but many more people are talking about genes and genetic testing these days.

Genetics is the domain of some very clever scientists and to fully understand this field would require a great deal of effort.  Often the cleverest people are the least able to explain things to the rest of us. Some equally clever researchers think that there will be so many possible autism genes that it is better to focus on the much smaller number of shared affected pathways.  I stand with the latter group.
Confused? He should be.

As is often the case, even a basic understanding of the principles does allow you to draw meaningful practical conclusions from the ever-expanding pool of research.

In most cases of idiopathic autism there is over/under expression of a very large number of genes.  Most of these genes do their job and produce perfectly functional proteins, just too much or too little. If you ran a test to sequence all those genes, most would come up as normal.  A variation in the expression in one gene can affect the expression of numerous others.

Ultimately what matters is, in each part of the body, which genes are turned on when they should be off and vice versa.

Some well-known syndromes result from missing copies, or extra copies, of certain genes.  These are called CNVs (Copy Number Variants). This is relatively easy to validate with existing tests.

The most common type of genetic variation among people is an SNP (single nucleotide polymorphism) and it represents a difference in a single DNA building block, called a nucleotide.  There are online database that log the effect of individual SNPs, which you can look up.

There are 12,932 known SNPs for the CACNA1C gene, which expresses the calcium ion channel Cav1.2. Ion channels are little gates in your cells that open and close as part of the signaling process that controls your body.  When these gates are faulty they might stay open, or stay shut, or you might just have too many of them; and you end up with a problem called a channelopathy. The Cav1.2 ion channel is known  to go wrong autism, bipolar and schizophrenia. If you want to give that gate a nudge to shut it, you use a channel blocker. A complication is that these same gates appear all over your body and a Cav1.2 blocker will affect them all. This is why some people get side effects.

There can be genetic mutations that result in a variation in the structure of the protein that is expressed.  This can be good (protective), or it can be bad.  This does not seem to underlie autism.

The great majority of research concerns the exome, a tiny part of the wider genome; the exome holds the information needed to express proteins.  The remaining 98% was thought to be “DNA junk”, but this appears not to be the case.  Variations in the 98% do matter, because they include things like silencers and enhancers that affect gene expression.  Only recently have scientists paid attention to the 98%; so much remains unknown.

Until recently, scientists thought that human diseases were caused mainly by changes in DNA sequence, infectious agents such as bacteria and viruses, or environmental agents. Now, researchers have demonstrated that changes in the epigenome also can cause, or result from, disease.

The epigenome consists of a record of the chemical markers attached to your DNA, like bookmarks that turn on or turn off particular genes.  These changes can be passed down via transgenerational epigenetic inheritance.  Changes to the epigenome result in changes to the function of the genome. 

Epigenomics/epigenetics, will become a vital part of efforts to better understand the human body and to improve health. Epigenomic maps may someday enable doctors to determine an individual's health status and predict a patient's response to therapies. 

Genetics can be made to sound bewildering complex:-

An analogy to the human genome stored on DNA is that of instructions stored in a book:

• The book (genome) would contain 23 chapters (chromosomes);
• Each chapter contains 48 to 250 million letters (A,C,G,T) without spaces;
• Hence, the book contains over 3.2 billion letters total;
• The book fits into a cell nucleus the size of a pinpoint;

 Or you can make it quite simple:-

·        The latest Boing 747 has 6,000,000 parts, and I expect many special extra ones in the new Air Force One variant.

·        Less than 25,000 different genes are needed to make a human and many of these appear not be essential.

·       As with jumbo jet you don't just need all the components, but you have to know where to put them and the correct sequence. Is the human equivalent of these instructions in the exome? or elsewhere in the genome?

I prefer simple.

There are different forms of genetic testing; simple ones test for a specific dysfunction, like Fragile-X.  This is relatively straight forward because the lab knows exactly where to look.

When you start looking for unknown dysfunctions the big risk is that you will find very many variances, the great majority of which have no relevance whatsoever.  You need to only consider the variances that are relevant.  Who decides which are relevant?  Beyond the very well-known risk genes, it becomes hugely subjective.

This interpretation is subjective because no one knows for sure the complete list of genes that could relate to autism.  It is likely to be a sub-set of the so-called "essential genes". The Simons Foundation suggest around 700 genes, but the list keeps growing. The AutsimKB database maintained by Peking University currently contains 3075 genes (99 syndromic autism related genes and 3022 non-syndromic autism related genes), 4964 Copy Number Variations (CNVs).

Some people will learn important things about their autism from today’s  genetic testing, but many will not.

Two things need to change, better data and better analysis.

I did check the analysis from one well-known US testing laboratory.  All I did was look at the genes they highlighted as being relevant to autism and then look at the supplementary list of variance present in one test, but that they considered irrelevant to autism.  Just using google I could find evidence that some of those irrelevant genes were actually potentially relevant.

Even if you find you have a flagged autism gene, this may or may not help you. 

If you have a defect in your mitochondrial DNA then you can say “for sure” that you have a particular type of mitochondrial disease and try and optimize your therapy.

If you have a variance in a gene associated with people with autism who remain non-verbal, you might be better off not knowing.  There is a case often quoted of the mother who made great efforts to get her child to talk, only to find later that a “non-verbal” mutation existed.  She might not have bothered had she made the genetic testing at a younger age.  I actually know a child diagnosed with a supposed “non-verbal” mutation and I think it is a really stupid diagnosis.  


A Future World

In a future time you might analyze the genome and epigenome of both parents and the child. This would be automatically be compared with the results from tens of millions of other people.  This would then reliably predict the possible dysfunctions that might develop in the child.  Having predicted the dysfunctions and the probability of them actually occurring,  a list of personalized therapies, some preventative, would be provided.

Conclusion

The science is not quite there yet. There is much work to be done on the exome, the wider genome, let alone the record of all those tags on it, which is the epigenome.

Current genetic testing can confirm known single gene autism syndromes.

Current genetic testing can identify known single gene metabolic and other disorders that can contribute to autism.

For idiopathic autism current genetic testing may, or may not, tell you anything that leads to a useful therapy.  Given the choice, take the best genetic testing available, just realize its limitations.

Now for the science:-

Understanding Genetics

There are some good introductions to genetics that are available for free.

Wikipedia gets quite complicated

This book is available for free:-

     Help Me Understand Genetics

  

DNA

DNA is the hereditary material in humans. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus, where it is called nuclear DNA, but a small amount of DNA can also be found in the mitochondria, where it is called mitochondrial DNA. 

The information in DNA is stored as a code made up of four chemical bases:

adenine (A), guanine (G), cytosine (C), and thymine (T).

Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences. 

DNA bases pair up with each other, A with T and C with G, to form units called base pairs.  Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. 

Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladder’s rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder. 

An important property of DNA is that it can replicate, or make copies of itself. 

Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell.

Mitochondrial DNA

Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. This genetic material is known as mitochondrial DNA. 

Mitochondria are structures within cells that convert the energy from food into a form that cells can use. Each cell contains hundreds to thousands of  mitochondria. 

Mitochondria produce energy through a process called oxidative phosphorylation. This process uses oxygen and simple sugars to create adenosine triphosphate (ATP), the cell’s main energy source. A set of enzyme complexes, designated as complexes I-V, carry out oxidative phosphorylation within mitochondria. 

In addition to energy production, mitochondria play a role in several other cellular activities. For example, mitochondria help regulate the self-destruction of cells (apoptosis). They are also necessary for the production of substances such as  cholesterol and heme (a component of hemoglobin, the molecule that carries oxygen in the blood). 

Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation. The remaining genes provide instructions for making molecules called transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are chemical cousins of DNA. These types of RNA help assemble protein building blocks (amino acids) into functioning proteins.

Genes

A gene is the basic physical and functional unit of heredity. Genes, which are made up of DNA, act as instructions to make molecules called proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases. The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes.

Every person has two copies of each gene, one inherited from each parent.

Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people.

Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each person’s unique physical features.

Genes are made up of DNA. Each chromosome contains many genes.

Chromosome

In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure.

Chromosomes are not visible in the cell’s nucleus—not even under a microscope - when the cell is not dividing.

In humans, each cell normally contains 23 pairs of chromosomes, for a total of 46.

We inherit 23 of our chromosomes from our mother (in the egg), and the other 23 from our father (in the sperm), so that we have 23 pairs of chromosomes, and therefore two copies of each gene.

Twenty-two of these pairs, called autosomes, look the same in both males and females. The 22 autosomes are numbered by size.

The 23rd pair, the sex chromosomes, differ between males and females. Females have two copies of the X chromosome, while males have one X and one Y chromosome.

Down syndrome is caused by possessing three copies of chromosome 21 instead of the usual two.

Fragile X syndrome is caused by a defect on chromosome 23, typically due to the expansion of the CGG triplet repeat within the Fragile X mental retardation 1 (FMR1) gene on the X chromosome.

Males with a full mutation display virtually complete penetrance and will therefore almost always display symptoms of FXS, while females with a full mutation generally display a penetrance of about 50% as a result of having a second, normal X chromosome. Females with FXS may have symptoms ranging from mild to severe, although they are generally less affected than males.

Do all gene mutations affect health and development?

No; only a small percentage of mutations cause genetic disorders—most have no impact on health or development. For example, some mutations alter a gene's DNA sequence but do not change the function of the protein made by the gene.

Often, gene mutations that could cause a genetic disorder are repaired by certain enzymes before the gene is expressed and an altered protein is produced.

Each cell has a number of pathways through which enzymes recognize and repair mistakes in DNA. Because DNA can be damaged or mutated in many ways, DNA repair is an important process by which the body protects itself from disease.

A very small percentage of all mutations actually have a positive effect. These mutations lead to new versions of proteins that help an individual better adapt to changes in his or her environment. For example, a beneficial mutation could result in a protein that protects an individual and future generations from a new strain of bacteria.

Because a person's genetic code can have a large number of mutations with no effect on health, diagnosing genetic conditions can be difficult. Sometimes, genes thought to be related to a particular genetic condition have mutations, but whether these changes are involved in development of the condition has not been determined; these genetic changes are known as variants of unknown significance (VOUS). Sometimes, no mutations are found in suspected disease related genes, but mutations are found in other genes whose relationship to a particular genetic condition is unknown. It is difficult to know whether these variants are involved in the disease.

The types of possible gene mutations

The DNA sequence of a gene can be altered in a number of ways. Gene mutations have varying effects on health, depending on where they occur and whether they alter the function of essential proteins. The types of mutations include:

Missense mutation

This type of mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene.

Nonsense mutation

A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all.

Insertion

An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by the gene may not function properly.

Deletion

A deletion changes the number of DNA bases by removing a piece of DNA. Small deletions may remove one or a few base pairs within a gene, while larger deletions can remove an entire gene or several neighboring genes.

The deleted DNA may alter the function of the resulting protein(s).

Duplication

A duplication consists of a piece of DNA that is abnormally copied one or more times. This type of mutation may alter the function of the resulting protein.

Frameshift mutation

This type of mutation occurs when the addition or loss of DNA bases changes a gene's reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids.

The resulting protein is usually nonfunctional. Insertions, deletions, and duplications can all be frameshift mutations.

Repeat expansion

Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. For example, a trinucleotide repeat is made up of 3-base-pair sequences, and a tetranucleotide repeat is made up of 4-base-pair sequences. A repeat expansion is a mutation that increases the number of times that the short DNA sequence is repeated. This type of mutation can cause the resulting protein to function improperly.

Change in the number of genes (CNVs)

People have two copies of most genes, one copy inherited from each parent. In some cases, however, the number of copies varies—meaning that a person can be born with one, three, or more copies of particular genes. Less commonly, one or more genes may be entirely missing. This type of genetic difference is known as copy number variation (CNV).

Copy number variation results from insertions, deletions, and duplications of large segments of DNA. These segments are big enough to include whole genes. Variation in gene copy number can influence the activity of genes and ultimately affect many body functions.

Copy number variation accounts for a significant amount of genetic difference between people. More than 10 percent of human DNA appears to contain these differences in gene copy number. While much of this variation does not affect health or development, some differences likely influence a person’s risk of disease and response to certain drugs.

Changes in the number of chromosomes

Human cells normally contain 23 pairs of chromosomes, for a total of 46 chromosomes in each cell. A change in the number of chromosomes can cause problems with growth, development, and function of the body's systems. These changes can occur during the formation of reproductive cells (eggs and sperm), in early fetal development, or in any cell after birth. A gain or loss of chromosomes from the normal 46 is called aneuploidy.

A common form of aneuploidy is trisomy, or the presence of an extra chromosome in cells. People with trisomy have three copies of a particular chromosome in cells instead of the normal two copies.

Down syndrome is an example of a condition caused by trisomy. People with Down syndrome typically have three copies of chromosome 21 in each cell, for a total of 47 chromosomes per cell.

Monosomy, or the loss of one chromosome in cells, is another kind of aneuploidy. People with monosomy have one copy of a particular chromosome in cells instead of the normal two copies. Turner syndrome is a condition caused by monosomy.  Women with Turner syndrome usually have only one copy of the X chromosome in every cell, for a total of 45 chromosomes per cell.

Rarely, some cells end up with complete extra sets of chromosomes. Cells with one additional set of chromosomes, for a total of 69 chromosomes, are called triploid. Cells with two additional sets of chromosomes, for a total of 92 chromosomes, are called tetraploid. A condition in which every cell in the body has an extra set of chromosomes is not compatible with life.

Changes in the structure of chromosomes

This type of change is usually associated with cancer.

Changes that affect the structure of chromosomes can cause problems with growth, development, and function of the body's systems. These changes can affect many genes along the chromosome and disrupt the proteins made from those genes.

Structural changes can occur during the formation of egg or sperm cells, in early fetal development, or in any cell after birth. Pieces of DNA can be rearranged within one chromosome or transferred between two or more chromosomes. The effects of structural changes depend on their size and location, and whether any genetic material is gained or lost. Some changes cause medical problems, while others may have no effect on a person's health.

Changes in chromosome structure include:

Translocations

A translocation occurs when a piece of one chromosome breaks off and attaches to another chromosome. This type of rearrangement is described as balanced if no genetic material is gained or lost in the cell. If there is a gain or loss of genetic material, the translocation is described as unbalanced.

Deletions

Deletions occur when a chromosome breaks and some genetic material is lost. Deletions can be large or small, and can occur anywhere along a chromosome.

Duplications

Duplications occur when part of a chromosome is copied (duplicated) too many times. This type of chromosomal change results in extra copies of genetic material from the duplicated segment.

Inversion

An inversion involves the breakage of a chromosome in two places; the resulting piece of DNA is reversed and re-inserted into the chromosome.

Genetic material may or may not be lost as a result of the chromosome breaks. An inversion that involves the chromosome's constriction point (centromere) is called a pericentric inversion. An inversion that occurs in the long (q) arm or short (p) arm and does not involve the centromere is called a paracentric inversion.

Isochromosomes

An isochromosome is a chromosome with two identical arms. Instead of one long (q) arm and one short (p) arm, an isochromosome has two long arms or two short arms. As a result, these abnormal chromosomes have an extra copy of some genes and are missing copies of other genes.

Dicentric chromosomes

Unlike normal chromosomes, which have a single constriction point (centromere), a dicentric chromosome contains two centromeres. Dicentric chromosomes result from the abnormal fusion of two chromosome pieces, each of which includes a centromere. These structures are unstable and often involve a loss of some genetic material.

Ring chromosomes

Ring chromosomes usually occur when a chromosome breaks in two places and the ends of the chromosome arms fuse together to form a circular structure. The ring may or may not include the chromosome's constriction point (centromere). In many cases, genetic material near the ends of the chromosome is lost.

Many cancer cells also have changes in their chromosome structure. These changes are not inherited; they occur in somatic cells (cells other than eggs or sperm) during the formation or progression of a cancerous tumor.

Changes in mitochondrial DNA

Mitochondria  are structures within cells that convert the energy from food into a form that cells can use. Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA (known as mitochondrial DNA or mtDNA). In some cases, inherited changes in mitochondrial DNA can cause problems with growth, development, and function of the body’s systems. These mutations disrupt the mitochondria’s ability to generate energy efficiently for the cell.

Conditions caused by mutations in mitochondrial DNA often involve multiple organ systems. The effects of these conditions are most pronounced in organs and tissues that require a lot of energy (such as the heart, brain, and muscles).

Although the health consequences of inherited mitochondrial DNA mutations vary widely, frequently observed features include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

Mitochondrial DNA is also prone to somatic mutations, which are not inherited. somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations. Because mitochondrial DNA has a limited ability to repair itself when it is damaged, these mutations tend to build up over time. A buildup of somatic mutations in mitochondrial DNA has been associated with some forms of cancer and an increased risk of certain age-related disorders such as heart disease, Alzheimer disease, and Parkinson disease. Additionally, research suggests that the progressive accumulation of these mutations over a person’s lifetime may play a role in the normal process of aging.

What does it mean to have a genetic predisposition to a disease?

A genetic predisposition (sometimes also called genetic susceptibility) is an increased likelihood of developing a particular disease based on a person's genetic makeup. A genetic predisposition results from specific genetic variations that are often inherited from a parent. These genetic changes contribute to the development of a disease but do not directly cause it. Some people with a predisposing genetic variation will never get the disease while others will, even within the same family.

Genetic variations can have large or small effects on the likelihood of developing a particular disease. For example, certain mutations in the BRCA1 or BRCA2 genes greatly increase a person's risk of developing breast cancer and ovarian cancer. Variations in other genes, such as BARD1 and BRIP1, also increase breast cancer risk, but the contribution of these genetic changes to a person's overall risk appears to be much smaller.

Current research is focused on identifying genetic changes that have a small effect on disease risk but are common in the general population. Although each of these variations only slightly increases a person's risk, having changes in several different genes may combine to increase disease risk significantly.

Changes in many genes, each with a small effect, may underlie susceptibility to many common diseases, including cancer, obesity, diabetes, heart disease, and mental illness.

In people with a genetic predisposition, the risk of disease can depend on multiple factors in addition to an identified genetic change. These include other genetic factors (sometimes called modifiers) as well as lifestyle and environmental factors.  Although a person's genetic makeup cannot be altered, some lifestyle and environmental  modifications (such as having more frequent disease screenings and maintaining a healthy weight) may be able to reduce disease risk in people with a genetic predisposition.

Genes direct the production of proteins

Most genes contain the information needed to make functional molecules called proteins. (A few genes produce other molecules that help the cell assemble proteins.)

The journey from gene to protein is complex and tightly controlled within each cell. It consists of two major steps: transcription and translation. Together, transcription and translation are known as gene expression. During the process of transcription, the information stored in a gene's DNA is transferred to a similar molecule called RNA (ribonucleic acid) in the cell nucleus.

Both RNA and DNA are made up of a chain of nucleotide bases, but they have slightly different chemical properties. The type of RNA that contains the information for making a protein is called messenger RNA (mRNA) because it carries the information, or message, from the DNA out of the nucleus into the cytoplasm.

Translation, the second step in getting from a gene to a protein, takes place in the cytoplasm. The mRNA interacts with a specialized complex called a ribosome, which "reads" the sequence of mRNA bases. Each sequence of three bases, called a codon, usually codes for one particular amino acid. (Amino acids are the building blocks of proteins.) A type of RNA called transfer RNA (tRNA) assembles the protein, one amino acid at a time. Protein assembly continues until the ribosome encounters a “stop” codon (a sequence of three bases that does not code for an amino acid).

The flow of information from DNA to RNA to proteins is one of the fundamental principles of molecular biology. It is so important that it is sometimes called the "central dogma.”

Genes turn on and off in cells

Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are repressed, or turned off. The process of turning genes on and off is known as gene regulation. Gene regulation is an important part of normal development. Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example. Gene regulation also allows cells to react quickly to changes in their environments. Although we know that the regulation of genes is critical for life, this complex process is not yet fully understood.

Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene’s DNA is transferred to mRNA). Signals from the environment or from other cells activate proteins called transcription factors. These proteins bind to regulatory regions of a gene and increase or decrease the level of transcription. By controlling the level of transcription, this process can determine the amount of protein product that is made by a gene at any given time.

The epigenome

DNA modifications that do not change the DNA sequence can affect gene activity. Chemical compounds that are added to single genes can regulate their activity; these modifications are known as epigenetic changes. The epigenome comprises all of the chemical compounds that have been added to the entirety of one’s DNA (genome) as a way to regulate the activity (expression) of all the genes within the genome. The chemical compounds of the epigenome are not part of the DNA sequence, but are on or attached to DNA (“epi-“ means above in Greek). Epigenomic modifications remain as cells divide and in some cases can be inherited through the generations. Environmental influences, such as a person’s diet and exposure to pollutants, can also impact the epigenome.

Epigenetic changes can help determine whether genes are turned on or off and can influence the production of proteins in certain cells, ensuring that only necessary proteins are produced. For example, proteins that promote bone growth are not produced in muscle cells. Patterns of epigenome modification vary among individuals, different tissues within an individual, and even different cells.

A common type of epigenomic modification is called methylation. Methylation involves attaching small molecules called methyl groups, each consisting of one

carbon atom and three hydrogen atoms, to segments of DNA. When methyl groups are added to a particular gene, that gene is turned off or silenced, and no protein is produced from that gene.

Because errors in the epigenetic process, such as modifying the wrong gene or failing to add a compound to a gene, can lead to abnormal gene activity or inactivity, they can cause genetic disorders. Conditions including cancers, metabolic disorders, and degenerative disorders have all been found to be related to epigenetic errors.

Scientists continue to explore the relationship between the genome and the

chemical compounds that modify it. In particular, they are studying what effect

the modifications have on gene function, protein production, and human health.

How geneticists indicate the location of a gene

Geneticists use maps to describe the location of a particular gene on a chromosome. One type of map uses the cytogenetic location to describe a gene’s position. The cytogenetic location is based on a distinctive pattern of bands created when chromosomes are stained with certain chemicals. Another type of map uses the molecular location, a precise description of a gene's position on a chromosome. The molecular location is based on the sequence of DNA building blocks (base pairs) that make up the chromosome.

Cytogenetic location

Geneticists use a standardized way of describing a gene's cytogenetic location.

In most cases, the location describes the position of a particular band on a stained chromosome:

        17q12

It can also be written as a range of bands, if less is known about the exact location:

        17q12-q21

The combination of numbers and letters provide a gene's “address” on a chromosome. This address is made up of several parts:

• The chromosome on which the gene can be found. The first number or letter used to describe a gene's location represents the chromosome. Chromosomes 1 through 22 (the autosomes) are designated by their chromosome number. The sex chromosomes are designated by X or Y.

• The arm of the chromosome. Each chromosome is divided into two sections (arms) based on the location of a narrowing (constriction) called the centromere. By convention, the shorter arm is called p, and the longer arm is called q. The chromosome arm is the second part of the gene's address. For example, 5q is the long arm of chromosome 5, and Xp is the short arm of the X chromosome.

• The position of the gene on the p or q arm. The position of a gene is based on a distinctive pattern of light and dark bands that appear when the chromosome is stained in a certain way. The position is usually designated by two digits (representing a region and a band), which are sometimes followed by a decimal point and one or more additional digits (representing sub-bands within a light or dark area). The number indicating the gene position increases with distance from  the centromere. For example: 14q21 represents position 21 on the long arm of chromosome 14. 14q21 is closer to the centromere than 14q22. Sometimes, the abbreviations “cen” or “ter” are also used to describe a gene's cytogenetic location. “Cen” indicates that the gene is very close to the  centromere. For example, 16pcen refers to the short arm of chromosome 16 near the centromere. “Ter” stands for terminus, which indicates that the gene is very close to the end of the p or q arm. For example, 14qter refers to the tip of the long arm of chromosome 14. (“Tel” is also sometimes used to describe a gene's location. “Tel” stands for telomeres, which are at the ends of each chromosome. The abbreviations “tel” and “ter” refer to the same location.)

The different ways in which a genetic condition can be inherited?

Some genetic conditions are caused by mutations in a single gene. These conditions are usually inherited in one of several patterns, depending on the gene involved:

Patterns of inheritance

Inheritance                   Description                                                                       Examples

pattern

Autosomal                     One mutated copy of the gene in each                      Huntington disease

Dominant                       cell is sufficient for a person to be affected               Marfan syndrome

by an autosomal dominant disorder. In

some cases, an affected person inherits the

condition from an affected parent.

.                                    In others, the condition may result from a new
                                      mutation in the geneand occur in people with
                                      no history of the disorder in their family

Autosomal                In autosomal recessive inheritance,                           Both cystic fibrosis

Recessive                copies of the gene in each cell have                          sickle

mutations. The parents of an individual with

an autosomal recessive condition each carry

one copy of the mutated gene, but they typically

do not show signs and symptoms of the condition.

Autosomal recessive disorders are typically

not seen in every generation of an affected

family.

X-linked                    X-linked dominant disorders are caused by               Fragile X

Dominant                  mutations in genes on the X chromosome,                syndrome

one of the two sex chromosomes in

each cell. In females (who have two X

chromosomes), a mutation in one of the two

copies of the gene in each cell is sufficient

to cause the disorder. In males (who have

only one X chromosome), a mutation in the

only copy of the gene in each cell causes

the disorder. In most cases, males experience

more severe symptoms of the disorder than

females. A characteristic of X-linked inheritance

is that fathers cannot pass X-linked traits to their

sons (no male-to-male transmission).

X-linked                    X-linked recessive disorders are also                         Hemophilia,

Recessive                caused by mutations in genes on the X                      Fabry disease

chromosome. In males (who have only

one X chromosome), one altered copy of

the gene in each cell is sufficient to cause

the condition. In females (who have two

X chromosomes), a mutation would have

to occur in both copies of the gene to

cause the disorder.

Because it is unlikely that females will have

two altered copies of this gene, males are

affected by X-linked recessive disorders

much more frequently than females. A

characteristic of X-linked inheritance is that

fathers cannot pass X-linked traits to their

sons (no male-to-male transmission).

Y-linked                   A condition is considered Y-linked if the                     Y chromosome

mutated gene that causes the disorder                      infertility, someis             located on the Y chromosome, one of                         cases of Swyer

the two sex chromosomes in each of a                     syndrome

male's cells. Because only males have a

Y chromosome, in Y-linked inheritance, a

mutation can only be passed from father to

son.

Codominant             In codominant inheritance, two different                    ABO blood group

versions (alleles) of a gene are expressed,

and each version makes a slightly different

protein. Both alleles influence the genetic trait

or determine the characteristics of the genetic

condition.

Mitochondrial            Mitochondrial inheritance, also known as                  Leber hereditary

maternal inheritance, applies to genes in                   optic neuropathy

mitochondrial DNA. Mitochondria, which                   (LHON)

are structures in each cell that convert

molecules into energy, each contain a

small amount of DNA. Because only

egg cells contribute mitochondria to the

developing embryo, only females can pass

on mitochondrial mutations to their children

Conditions resulting from mutations in

mitochondrial DNA can appear in every

generation of a family and can affect both

males and females, but fathers do not pass

these disorders to their daughters or sons.

Many health conditions are caused by the combined effects of multiple genes or by interactions between genes and the environment. Such disorders usually do not follow the patterns of inheritance described above. Examples of conditions caused by multiple genes or gene/environment interactions include heart disease, diabetes, schizophrenia, and certain types of cancer.

Disorders caused by changes in the number or structure of chromosomes also do not follow the straightforward patterns of inheritance listed above.

Other genetic factors sometimes influence how a disorder is inherited.

Reduced penetrance and variable expressivity

Reduced penetrance and variable expressivity are factors that influence the effects of particular genetic changes. These factors usually affect disorders that have an autosomal dominant pattern of inheritance, although they are occasionally seen in disorders with an autosomal recessive inheritance pattern.

Reduced penetrance

Penetrance refers to the proportion of people with a particular genetic change (such as a mutation in a specific gene) who exhibit signs and symptoms of a genetic disorder. If some people with the mutation do not develop features of the disorder, the condition is said to have reduced (or incomplete) penetrance.

Reduced penetrance often occurs with familial cancer syndromes. For example, many people with a mutation in the BRCA1 or BRCA2 gene will develop cancer during their lifetime, but some people will not. Doctors cannot predict which people with these mutations will develop cancer or when the tumors will develop.

Reduced penetrance probably results from a combination of genetic, environmental, and lifestyle factors, many of which are unknown. This phenomenon can make it challenging for genetics professionals to interpret a person’s family medical history and predict the risk of passing a genetic condition to future generations.

Variable expressivity

Although some genetic disorders exhibit little variation, most have signs and symptoms that differ among affected individuals. Variable expressivity refers to the range of signs and symptoms that can occur in different people with the same genetic condition. For example, the features of Marfan syndrome vary widely— some people have only mild symptoms (such as being tall and thin with long, slender fingers), while others also experience life-threatening complications involving the heart and blood vessels. Although the features are highly variable, most people with this disorder have a mutation in the same gene (FBN1).

As with reduced penetrance, variable expressivity is probably caused by a combination of genetic, environmental, and lifestyle factors, most of which have not been identified. If a genetic condition has highly variable signs and symptoms, it may be challenging to diagnose.

Gene therapy

Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

• Replacing a mutated gene that causes disease with a healthy copy of the gene.

• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.

• Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently only being tested for the treatment of diseases that have no other cures.

How does gene therapy work?

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body

Genetic Testing Methods

If you made it through the above information you may be interested in how it is possible to detect these genetic variances.

We are looking for two distinct types of variances, SNPs (Single-Nucleotide Polymorphism) and CNVs (Copy Number variants).

Microarray-based genotyping vs Whole Exome/Genome Sequencing

There are three general types of testing that you might encounter in trying to achieve a biological diagnosis of a person’s autism.

The simplest and cheapest is microarray genotyping, also known as chromosomal microarray CMA .  This tests the prevalence of a very large number of known suspect DNA sequences that have been programed into the machine.  CMA is often suggested as the first tier test for individuals with developmental disabilities, intellectual disabilities, autism spectrum disorders, or multiple congenital anomalies.  This is a good method if you know what variances you are looking for.

Whole Exome Sequencing (WES) established the exact nucleotide sequences of DNA at the thousands of exon loci tested.  This test used to be very expensive, but is now quite widely available in the United States, both for research and commercially.

Whole Genome Sequencing (WGS) looks at the entire genome, rather than the much smaller exome.  This test is available, but normally with the caveat “for research purposes”. This should be the holly grail of genetic testing.

The cost of all types of genetic testing continues to fall.

The tests are only as good as the interpretation of their results.

If you live in the US, you can join SPARK for free. This is another initiative of the Simons Foundation, this time to collect genetic material from people affected by autism. They aim to collect saliva samples from 50,000 people with autism, and the their families.  They will then conduct Whole Exome Sequencing (WES).

                          https://sparkforautism.org/portal/page/about-spark/



A more ambitious project is MSSNG, which Google backed project to analyse the entire genome in 10,000 people with autism.

https://www.mss.ng/#